DCIS
DCIS contain numerous, recurrent genetic abnormalities. Large chromosome alterations or aneuploidy (often detected by CGH) [4, 5, 6, 7, 8, 9, 10, 11] , oncogene amplification [12, 13, 14] , AI/LOH [8, 15, 16, 17, 18, 19] , expression profiles [20] and centrosome abnormalities [21, 22] can be indistinguishable from those in invasive ductal cancers (IDC). DCIS are also cytologically or immunohistochemically similar to IDC [12, 23, 24] . These observations, combined with the lesions' frequent presence alongside IDC, lead to the current view that DCIS are the immediate, although non-obligate, precursors of IDC.

Abnormalities of chromosomes 1q, 5p, 6q, 8q, 17q, 19q, 20p, 20q, Xq (generally gains), and of 2q, 5q, 6q, 8p, 9p, 11q, 13q, 14q, 16q, 17p, 22q (generally losses), as well as amplification of the chromosome regions that include the CMYC (c-myc), CCND1 (cyclin D1), and ERBB2 (Her2/neu) oncogenes are noted commonly in DCIS. Low-grade lesions often have 1q and 16q alterations, whereas high-grade lesions tend to have changes in 13q, 17q and 20q. Similar findings in low and high-grade invasive carcinomas have led to speculation that low and high-grade lesions develop through distinct pathways, rather than by "dedifferentiation" or clonal evolution [25, 26] . Thus, low-grade DCIS would be a precursor of a low-grade invasive carcinoma or a tubular or tubulo-lobular cancer. High grade DCIS would be a precursor of a high-grade invasive carcinoma. Support for this model comes from gene microarray expression data showing that different tumor grades, but not tumor stages, are associated with distinct gene expression signatures [20]. In addition, centrosome abnormalities are associated with high-grade DCIS and with chromosome instability or aneuploidy [21].

Taken together, findings indicate that many of a breast cancer's genetic abnormalities are already present in preinvasive lesions. The order of appearance of different genetic alterations is controversial; some posit mutation, rearrangement, loss or amplification of particular genes leading to a growth advantage in the affected cell [27, 28] , while others posit aneuploidy as the initiating event [29, 30] . It may not be an either-or situation. Regardless, if so many recurrent abnormalities exist in DCIS, then it implies that precursor lesions exist. These precursors would contain fewer abnormalities, and these abnormalities would be critical to initial stages of tumorigenesis.

Precursors of DCIS
The most likely candidate precursors include the proliferative lesions, particularly simple or usual ductal hyperplasia (DH) and atypical ductal hyperplasia (ADH). Clinically, these lesions are considered benign, but epidemiologic studies indicate that their diagnosis is associated with increased risk of subsequent breast cancer [31, 32, 33, 34, 35, 36, 37] . They are found alongside breast cancers in surgical specimens, and are present in greater numbers in breasts with cancer or at high risk of developing cancer than in controls [38, 39, 40, 41, 42, 43] . A fraction of DH and ADH demonstrate genetic abnormalities also detected in either in situ or invasive cancers. Particularly fruitful have been studies of AI/LOH [15, 19, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53] and of cytogenetic abnormalities [54, 55, 56, 57] both of which can be performed on archived samples. These studies have generated the prevailing model of ductal breast carcinogenesis as a sequential progression from normal to DH to ADH to in situ to invasive malignancy. However, evidence for the early stages of this model is limited and the biological relationship of DH, ADH and DCIS remains far from clear.

ADH
ADH are excellent candidate malignant precursors. They resemble DCIS histologically, and they can contain similar AI/LOH [19, 44, 46] and cytogenetic abnormalities [55, 56] . Studies indicate that monoclonal alterations are present in 25-50% of lesions [15, 44, 58, 59, 60, 61] , often at 8p, 16p, and 17q [44, 58] . However, only 15% of women diagnosed with ADH eventually develop a breast malignancy, so these lesions are certainly not committed precursors and they may be genetically heterogeneous.

Several studies have examined molecular abnormalities in co-existing DH, ADH and or cancers. AI/LOH [19, 44, 45, 53, 62] , Her2/Neu amplification [63], gene hypermethylation [64], cytogenetic abnormalities [54, 55, 56] , and RNA expression [20] have been investigated. Usually, these studies describe a complicated and genetically heterogeneous picture, with both concordant and discordant alterations present in proliferative lesions and cancers. Although each study examines only a small number of molecular features or samples, overall, they support the model of ADH as a non-obligate cancer precursor. The extent of genetic similarity between ADH and cancers is uncertain. In addition, it not known whether any alterations are present consistently in proliferative lesions but not cancers. Such discordant alterations could suggest loci whose abnormalities are not causative but might be arresting neoplastic development. In sum, a more complete picture of the genetic relationship between hyperplastic lesions and cancers is needed.

To this end, we conducted a comprehensive investigation of AI/LOH in microdissected hyperplastic lesions and co-existing cancers. Using 21 markers on 10 chromosome arms, we characterized 106 lesions (13 DH, 45 ADH, 30 DCIS and 18 IDC) from 17 independent breast-cancer containing specimens. The data were analyzed using statistical programs weighting for different numbers of lesions per case, variable heterozygosity per marker, and different numbers of markers per arm. Our preliminary results indicate that AI/LOH in ADH may predict characteristics of co-existing cancers. Since genetic characteristics of breast cancers appear to correlate with clinical outcome [65, 66, 67] , identifying predictive markers in ADH could have considerable implications.

ALH/LCIS
In contrast to the ductal proliferative lesions, the lobular proliferative lesions ALH and LCIS have been considered to be markers of risk rather than premalignant lesions themselves. This role will have to be reconsidered in light of several large retrospective studies indicating increased ipsilateral risk of breast cancer in patients diagnosed with ALH or LCIS [68, 69, 70] . Fewer molecular studies have been reported on preinvasive lobular proliferations than ductal proliferations, but the results are all consistent with the conclusion that at least a proportion of ALH and LCIS represent clonal neoplasms that are potential cancer precursors [71, 72, 73] . Using CGH, a direct comparison between LCIS and DCIS suggests that the genetic changes in LCIS resemble those in well-differentiated DCIS, with frequent 1q and 16q abnormalities [74], but whether this finding applies to invasive cancers awaits further investigations.

DH/simple hyperplasia/hyperplasia of the usual type (HUT)
Only 5% of women diagnosed with DH eventually develop invasive carcinomas. While the presence of DH is considered a risk factor for subsequent cancer development, these lesions are clearly not committed precursors. Small proportions of hyperplastic lesions (0-15%) contain either LOH [19, 47, 50] or cytogenetic abnormalities [54, 55, 57, 75] suggesting that they are monoclonal. However, their role as precursors of cancers, or even of ADH, is uncertain. Because the number of abnormalities per DH, and the number of DH that have been investigated, are smaller than DCIS or ADH, recurrent sites of chromosomal abnormalities in DH are hard to identify with confidence.

Recent immunohistochemical (IHC) and immunofluoresence studies suggest that DH may not be part of the premalignant continuum, but instead are derived from mammary progenitor cells [75, 76] (see below). Using monoclonal antibodies thought to distinguish progenitor cells and differentiated luminal cells, DH were shown to consist of a mixture of glandular cells at different stages of glandular differentiation. Some were progenitors (cytokeratin (CK) 5+ and CK8/18-), others were transitional cells at an intermediate stage (CK5+ and CK8/18+), others were differentiated (CK5- and CK8/18+). The outer layer of the duct appeared to consist of normal myoepithelium (smooth muscle actin (SMA)+). Thus, DH could represent the gland's normal process of regeneration or response to hormonal stimulation. Additional studies, using complementary methods, should clarify whether any DH are true cancer precursors.

Normal epithelium
Given the number of genetic abnormalities in proliferative lesions, it is perhaps not surprising that clonal abnormalities, specifically AI/LOH, have been identified in normal-appearing epithelium [50, 77, 78, 79, 80, 81, 82] . AI/LOH, often at loci implicated in breast carcinogenesis, can be found in seemingly normal breast epithelium from women with and without breast cancer, and in tissue both adjacent to and distant from primary tumors. In breasts without malignant changes, genetic alterations in normal cells are infrequent, and perhaps random [75, 78] . In breasts with malignant change, genetic alterations in normal-appearing tissue are sometimes concordant with the changes in adjacent carcinomas [77, 83] , but other studies find that concordance is low [81].

Nothing is known about the settings or mechanisms leading to the appearance of these abnormalities, or whether they are biologically meaningful or predictive of outcome. A major advance in our knowledge would be to determine whether the genetically aberrant clones existing in histologically normal epithelium represent "background" abnormalities, premalignant lesions, or markers of increased risk.

We [78] and others [82] have shown that as risk of cancer increases, normal breast epithelium tends to have an increased number of genetic alterations. LOH in normal breast TDLUs may predict for local recurrence [80], perhaps by indicating persistence of malignant tissue not removed during surgery. Normal epithelium from BRCA1 or BRCA2 germline mutation carriers, compared to non-carriers, expresses predominantly the PR-A isoform and has altered expression of estrogen responsive proteins [84] (but not of ER itself). Finally, the abnormalities in normal epithelium appear to affect small chromosome regions, rather than whole chromosomes [81]. This suggests that processes leading to segmental abnormalities (such as DNA damage recognition or repair) would occur first, and abnormalities leading to aneuploidy (such as chromosome instability) would occur later.

Stroma
The important role of the tumor microenvironment, which includes the surrounding stromal tissue, in the development of epithelial cancers has been demonstrated in vitro and in model systems (for reviews see [85, 86] ). These data have generated the hypothesis that the stroma normally suppresses growth of malignant epithelium, but that stromal abnormalities could limit this suppression thereby permitting malignant development, especially invasion. Consistent with this hypothesis (although other interpretations are possible), in vivo studies report alterations either in the DNA (AI/LOH, mutations) or the protein expression (IHC) of stroma surrounding breast malignancies. They demonstrate abnormalities of important growth related pathways (for instance, PTEN, p53, the Wnt-APC-beta catenin pathway) that are usually not concordant with abnormalities in epithelium [83, 87, 88, 89] . Analyses of stromal tissue surrounding early epithelial breast cancer precursors (normal-appearing, hyperplastic or DCIS) independently or in parallel with the epithelial compartment have not been reported.

Mammary progenitor cells
The resting human mammary gland consists of a branching ductal system and terminal ductal lobulo-alveolar unit (TDLU) or lobule, which are the functional unit of the breast. Each lobule is lined by a layer of luminal epithelial cells surrounded by a basal layer of myoepithelial cells. The existence of mammary gland stem cells was first demonstrated in mouse studies. These studies showed that fragments of epithelium isolated from different regions of a mammary gland, at different stages of post-natal development, could be transplanted into a cleared mammary fat pad and were capable of generating fully functional mammary epithelial outgrowths containing ducts, lobules and myoepithelial cells [90].

Until recently, information about human breast mammary stem cells has been limited. X-inactivation analyses [91, 92] suggested that entire lobules (large ducts and TDLUs) are monoclonal in origin, i.e., are derived from a single stem cell, although the "patch size" was not certain. As a consequence, any lesion arising in a TDLU would appear monoclonal, regardless of whether it arose from a stem cell or a differentiated cell. Human breast cells obtained from reduction mammoplasties have been cultured after separating luminal (CK 18/19 [+]) from myoepithelial (SMA [+]) cells. The luminal cells could produce myoepithelial cells, but not vice versa [93]. Recently, a subpopulation of luminal cells was identified that could generate themselves, myoepithelial cells and TDLU-like structures [94]. These results suggest that precursor cells exist in the luminal compartment. Other studies have defined putative mammary stem cells using other markers: as CK5+ and CK 8/18- [95], as CK19+ [94, 96] , as p21+ [97] or as a "side population" of cells able to efflux Hoechst 33342 dye [98]. A current model is that quiescent stem cells (however they are defined) are scattered throughout the normal lobular breast epithelium [97]. The relation of these stem cells to the genetically aberrant clones detected in some TDLU [81] is unknown.

The relation between stem cells and carcinogenesis is potentially of great importance [99]. Normal stem cells and cancers share the capacity for self-renewal and the ability to differentiate [100]. Conceivably, mutation of the stem cell could lead to cancer. Like putative stem cells, most (though not all) breast tumors express markers of luminal, not myoepithelial cells [101, 102, 103, 104, 105] . This is compatible with early studies suggesting that most breast cancers arose from TDLUs [106]. In rats, carcinogen-induced tumorigenesis appears to target an early progenitor or stem cell [107]. A recent report describes the transcriptional profile of a candidate human mammary stem cell [108]; how this compares to cancers' profiles will be of great interest. On the other hand, human cancers are genetically, phenotypically and clinically diverse. In transgenic mice, the particular oncogenic stimulus contributes to morphologically and genetically distinct tumors that result [109]. Thus, the highly heterogeneous nature of human breast cancer could be due in part to interactions between when in development the stem cell acquires a mutation, and what is the oncogenic stimulus.

The future
The existing model of a linear or simple sequential pathway to carcinogenesis may need refinements. New information, often obtained using new techniques, regarding the roles of genetic instability, mammary progenitor cells and tumor-stroma interactions, among others, should be considered. Incorporating the diverse results will be challenging, but knowledge of the genetic abnormalities critical in the early stages of tumorigenesis in vivo is important for understanding and estimating cancer risk, for identifying premalignant lesions destined to progress, for selection of potential targets for novel chemopreventive agents, and for increased understanding of cancer etiology.

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